Dataset Information

APPLICATION OF A NOVEL PEPTIDE SPECTRAL LIBRARY USING SWATH ANALYSIS FOR THE QUANTITATION OF PROTEINS IN EX-DIAGNOSTIC CLINICAL SERUM SAMPLES FROM SHEEP

ABSTRACT: This project applies SWATH approach to analyse ex-diagnostic sheep serum samples from actual clinical cases submitted to The University of Queensland's School of Veterinary Science, Veterinary Laboratory Services (VLS) compared with serum of normal sheep.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Ovis Aries

TISSUE(S): Blood Serum

SUBMITTER: Saul Chemonges

LAB HEAD: Dr Pawel Sadowski

PROVIDER: PXD005077 | Pride | 2022-11-15

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	20160805_SC_SWATH_i502_5uL.wiff	Wiff
	20160805_SC_SWATH_i502_5uL.wiff.scan	Wiff
	20160805_SC_SWATH_i503.wiff	Wiff
	20160805_SC_SWATH_i503.wiff.scan	Wiff
	20160805_SC_SWATH_i504.wiff	Wiff

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Project description:Several proteomics studies have been performed in the past in order to define the mammalian peroxisomal proteome. During the last decade, it has as well become evident, that a significant number of proteins is shared between several subcellular compartments and that specialized proteins subsets are constituents of membrane contact zones, which bridge two adjacent organelle to allow metabolite exchange. In this respect, a number of proteins, which have been considered as mere contaminants in previous studies, might be indeed proteins, which are to a minor extent indeed truly associated with peroxisomes. Ongoing technical improvements in mass spectrometry (MS) allow to identify and to quantify the enrichment of such less abundant proteins in individual fractions. Accordingly, this study reassessed the proteome of mouse liver peroxisomes by parallel isolation of peroxisomes from a mitochondria- and a microsome-enriched pre-fraction combining density gradient centrifugation with a SWATH-MS quantitative proteomics approach to unveil novel peroxisomal or peroxisome-associated proteins. In total, 1071 proteins from the MS-samples were identified in amounts, which allowed their quantification in order to assess their enrichment in either high density peroxisomal or low-density gradient fractions, containing the bulk of organelle material. Combining the data from isolations from the mitochondria- and microsome-enriched prefractions allowed to identify specific protein clusters characteristic for mitochondria, the ER and peroxisomes. Among the proteins, which were significantly enriched in the peroxisomal cluster, were several proteins, which were not previously associated with the organelles implying that they are novel peroxisomal candidates. In order to validate the organelle proteomics strategy, five of the candidates were selected for further colocalization studies. The peroxismal import of two candidates, HTATIP2 and PAFAH2, which contain potential peroxisome targeting sequences 1, could be confirmed by overexpression in HepG2 cells. Two other candidates, SAR1B and PDCD6, which are known from ER exit sites, did not directly colocalize with peroxisomes but were found to reside at ER sites which often surrounded peroxisomes. Hence, both proteins might concentrate at peroxisome-ER membrane contact sites, which might be co-purified with peroxisomes. Intriguingly, the last candidate, OCIA domain-containing protein 1, was previously described to decrease mitochondrial branching and network formation. In this work, we not only validate its secondary peroxisomal localization but also observed a reduction in peroxisome numbers in response OCIAD1 expression. Hence, OCIAD1 appears to be a novel protein, which has an impact on both mitochondrial and peroxisomal maintenance.

Dataset Information

APPLICATION OF A NOVEL PEPTIDE SPECTRAL LIBRARY USING SWATH ANALYSIS FOR THE QUANTITATION OF PROTEINS IN EX-DIAGNOSTIC CLINICAL SERUM SAMPLES FROM SHEEP

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